Enhancement of dendritic cell immunotherapy by recalling antigens for hepatocellular carcinoma in mice.

Background: The therapeutic efficacy of dendritic cell (DC)-immunotherapy for large hepatoma in mice is unsatisfactory. Materials & methods: DC-based immunotherapy was used to treat Hepa1-6 tumors measuring 6 ± 1 mm in diameter, enhanced by boosting tumor antigens. Results: CD4+ and CD8+ T-cells were contracted and transformed into memory phenotypic cells after DC-based vaccination. When T-cells were re-stimulated, T-cells obtained from mice boosted by tumor antigen injection showed highest proliferation capacity. When mice with large tumors were treated, DC-based vaccination boosted by tumor antigen and an additional DC-infusion yielded curative rates of 50% and 23.1%, respectively. Conclusion: DC vaccination induced effector memory cells. Antigen presentation recalled by DC or tumor antigens increased the curative rate in mice with large tumors.

Hepatocellular carcinoma is the most common liver malignancy and is often found at advanced stage. Immune checkpoint inhibitor combined with a molecular targeting agent is a new strategy for the treatment of advanced hepatocellular carcinoma and yields 30% of objective response rate. However, we still need another treatment for the patients who are not responsive to immune checkpoint inhibitor combined with a molecular targeting agent. Dendritic cell-based immunotherapy is one of the treatments for advanced hepatocellular carcinoma. In this animal study, dendritic cells can activate T-lymphocytes to kill cancer cells. Dendritic cells can also induce memory T-lymphocytes, which can be reactive by boost tumor antigens and increase therapeutic efficacy. This treatment strategy, dendritic cell infusion followed by tumor-antigen injection, can be translated into clinical practice in the future.

Immunological discrepancy in aged mice facilitates skin allograft survival.

More and more aged people are undergoing organ transplantation. Understanding aging effects on immunity will be helpful for post-transplantation care and adjustment of immunosuppressants for aged recipients. A mouse model, using C3H mice as donors and aged/young C57BL/10J mice as recipients, was employed to study aging effects on immunity. The results showed that frequency of myeloid-derived suppressor cells (MDSC) and level of TGF-ß was higher in aged mice than in young mice (4.4 ± 1.4% versus 1.6 ± 1.1%, p = 0.026 for MDSC; 21.04 ± 3.91 ng/ml versus 15.26 ± 5.01 ng/ml, p = 0.026 for TGF-ß). In vivo, skin allograft survived longer on the aged than on young mice (19.7 ± 5.2 days versus 11.9 ± 4.1 days, p = 0.005). When entinostat was applied to block MDSC, the survival of skin allografts on aged mice was shorten to 13.5 ± 4.7 days which was not different from the survival on young mice (p = 0.359). In conclusion, allogeneic immunity was different in aged from young mice in high frequency of MDSC and high serum level of TGF-ß. Blocking the function of MDSC reversed the low immunity in aged mice and caused skin allograft rejection similar to young recipients.

Stem cell factor produced by tumor cells expands myeloid-derived suppressor cells in mice.

Immunotherapy is a novel treatment approach for cancers; however, its therapeutic effects are impeded by myeloid-derived suppressor cells (MDSCs). This study aimed to determine how MDSCs are expanded in cancer hosts. MDSCs were positive for Gr-1 and CD11b. Hepa1-6 hepatoma cells, EL4 lymphoma cells, and mice bearing Hepa1-6 hepatoma or lymphoma were examined. Following the inoculation of Hepa1-6 cells into the flanks of mice, a linear correlation was evident between the frequency of MDSCs in the spleen and tumor sizes. MDSC numbers diminished gradually and returned to the normal level within 3 weeks if the tumors were excised. To identify the cytokines produced by tumor cells that allowed expansion of MDSCs, cytokines in Hepa1-6 cell culture medium and murine serum were examined using a cytokine array. Stem cell factor (SCF) was implicated as the relevant cytokine. When recombinant SCF was added to the spleen cell culture medium, MDSC expansion could occur. In the presence of c-kit blockade, this effect of SCF was partially reversed. In conclusion, MDSCs can be expanded in tumor cells in a process that involves SCF released by tumor cells.

Down-regulation of metabolic proteins in hepatocellular carcinoma with portal vein thrombosis.

BACKGROUND: Hepatocellular carcinoma is an aggressive malignancy with poor prognosis and easy to recur even the tumor is totally removed by surgery. Portal vascular invasion is one of the major factors contributing to tumor recurrence and poor prognosis. However, why hepatocellular carcinoma is easy to grow into vessels is unclear. METHODS: Surgical specimens from seven hepatocellular carcinoma patients with portal vein thrombosis and seven patients without vascular invasion were utilized to analyze protein expression by proteomic technique. The proteins in the tumors were separated by 2-dimensional electrophoresis. Protein patterns in the gels were recorded as digitalized images. The differences of expression in hepatocellular carcinoma with or without portal vein thrombosis were identified by mass spectrometry. RESULTS: Clinically, the tumors with portal vein thrombosis were larger than those without portal vein thrombosis. The median survival time for the patients with portal vein thrombosis was much shorter [4 (ranged 2.5-47) vs. 53 (ranged 33-85) months, p = 0.002]. By analyzing the protein expression in cancer tissues with or without portal vein thrombosis, the differences of protein expression were mainly metabolic enzymes. Carbonic anhydrase I, betaine-homocysteine S-methyltransferase 1, fumarate hydratase, isovaleryl-CoA dehydrogenase, short-chain specific acyl-CoA dehydrogenase and arginase-1 were all down-regulated in the tumors with portal vein thrombosis. CONCLUSION: Metabolic enzymes and cytosol carbonic anhydrases were downregulated in hepatocellular carcinoma with portal vein thrombus. The deficiency of metabolic enzymes and cytosol carbonic anhydrases may alter cellular metabolisms and acid-base balance in hepatocellular carcinoma, which may facilitate to invade portal vein.

Hepatic stellate cells promote the differentiation of embryonic stem cell-derived definitive endodermal cells into hepatic progenitor cells.

AIM: Hepatic non-parenchymal cells are well known to be capable of providing an important microenvironment and growth factors for hepatic regeneration, but their capacity for directing embryonic stem cells (ESC) toward hepatocytes remains to be assessed. Thus, this study aims to investigate the role of hepatic stellate cells (HSC), the major type of hepatic non-parenchymal cells, in the differentiation of ESC as well as exploring the potentiality of ESC in regeneration medicine for cell-based therapy. METHODS: A two-step differentiation procedure that utilized the capability of HSC to regulate proliferation and differentiation of hepatocytes was used to develop an approach for directing the differentiation of ESC towards hepatic progenitor cells. Mouse ESC were cultivated in a serum-free medium containing Activin A and fibroblast growth factor to generate definitive endodermal cells characterized by the CXCR4 cell-surface marker. After 6-8 days in culture, approximately 60% of the differentiated cells expressed CXCR4, and more than 90% of the CXCR4 positive cells could be recovered by cell sorting. The purified CXCR4 positive cells were co-cultured with mouse HSC as feeder cells in basal medium without additional hepatocyte growth factors. Differentiation was complete after 10-12 days of co-culture, and hepatic progenitor cell markers such as α-fetoprotein (afp) and albumin (alb) were detected in the terminally differentiated ESC. CONCLUSION: These results show that HSC provide an appropriate microenvironment and pivotal growth factors for generation of hepatic progenitor cells from ESC-derived definitive endodermal cells, and suggest that this approach possibly allows for hepatic differentiation of ESC imitating the process of hepatic regeneration.

The impact of CD4+ CD25+ T cells in the tumor microenvironment of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common liver cancer. Therapeutic results are usually unsatisfactory because liver tumors recur often. Immunologic factors may be related to the recurrence of HCC; however, this possibility is mentioned only rarely. METHODS: Thirty HCC patients undergoing hepatectomies were divided into 3 groups according to the diameters of their HCCs: group A (n = 8), diameter ≤3 cm; group B (n = 8), diameter >3 cm and ≤5 cm; and group C (n = 14), diameter >5 cm. T-lymphocytes from peripheral blood, nontumor liver tissue, and the HCC were analyzed. RESULTS: The percentage of CD25+ in the CD4+ T cells did not differ between the peripheral blood and the nontumor liver tissue among the 3 groups. CD25+ cells were increased in the tumor tissue in group C patients (range, 6-41%; median, 22.9%; P = .003), compared to group A patients. The percentage of CD25+ in the CD4+ T cells in tumor tissue was positively correlated with tumor sizes (r = 0.556). These CD4+ CD25+ lymphocytes produced transforming growth factor-ß and interferon-γ but not interleukin-10, and were anergic to plate-coated monoclonal antibodies (anti-CD3/anti-CD28). The characteristics of these antibodies were comparable to those of regulatory T cells. When the infiltration lymphocytes including CD4+ CD25+ T cells were added to the mixed lymphocyte reaction activated by autologous tumor lysate-pulsed dendritic cells, the proliferation of lymphocytes was inhibited. CONCLUSION: The increase of CD4+ CD25+ T cells in the tumor microenvironment correlates with tumor sizes. These CD4+ CD25+ regulatory T cells appeared to suppress the immune response activated by dendritic cells.